This year we have continued to identify novel antibodies and engineered antibody domains against cancer-related proteins. These antibodies are tested for their activity against cancer cells in vitro and some of them are also used for conjugation with nanoliposomes and for development of novel approaches for multiple site-specific conjugations of bioactive molecules to antibody fragments. We have also developed several large libraries containing billions of dAbs and nAbs which can be used as a source of binders to various targets. The major accomplishments are summarized below. 1) We have proposed to use isolated CH2 domains as scaffolds for construction of libraries containing diverse binders that could also confer some effector functions (nAbs). However, previous work has shown that an isolated murine CH2 domain is relatively unstable to thermally induced unfolding. To explore unfolding mechanisms of isolated human CH2 and increase its stability &amp;#947;1 CH2 was cloned and a panel of cysteine mutants was constructed. Human &amp;#947;1 CH2 unfolded at a higher temperature (Tm = 54.1C, as measured by circular dichroism) than that previously reported for a mouse CH2 (41C). One mutant (m01) was remarkably stable (Tm = 73.8C). Similar results were obtained by differential scanning calorimetry. This mutant was also significantly more stable than the wild-type CH2 against urea induced unfolding (50% unfolding at urea concentration of 6.8 M vs 4.2 M). The m01 was highly soluble and monomeric. The existence of the second disulfide bond in m01 and its correct position were demonstrated by mass spectrometry and nuclear magnetic resonance spectroscopy, respectively. The loops were on average more flexible than the framework in both CH2 and m01, and the overall secondary structure was not affected by the additional disulfide bond. These data suggest that a human CH2 domain is relatively stable to unfolding at physiological temperature, and that both CH2 and the highly stable mutant m01 are promising new scaffolds for the development of therapeutics against human diseases. 2) Several large (size 1010) phage-displayed libraries of engineered CH2 domains were constructed and are being tested for selection of binders. We are also expressing human neonatal Fc receptor (FcRn) to test binding of some of the engineered CH2 domains with the specific aim to generate nAbs that can bind specifically to a cancer-related protein and simultaneously to the FcRn. 3) We have previously constructed a large (size 2.5 1010) dAb library by grafting human antibody heavy chain complementarity determining regions (CDRs) 2 and 3 (H2s, H3s) into their cognate positions in a human heavy chain variable domain (VH) scaffold and mutagenizing the CDR1 (H1). High-affinity binders against some antigens were selected from this library but panning against others was not very successful likely due to limited diversity. We have hypothesized that by grafting highly variable, both in length and composition, human CDRs into non-cognate positions, the dAb library diversity could be significantly increased and the library would allow for more efficient selection of high-affinity antibodies against some targets. To test this hypothesis we designed a novel type of dAb library containing CDRs in non-cognate positions. It is based on our previous library where H1 was replaced by a library of human light chain CDR3s (L3s) thus combining three most diversified fragments (L3, H3 and H2) in one VH scaffold. This large (size 1010) phage-displayed library was highly diversified as determined by analyzing the sequences of randomly selected clones. The new library could be used not only for selection of such dAbs thus complementing existing libraries but also as a research tool for exploration of the mechanisms determining folding and stability of human antibody domains. 4) Novel high-affinity dAbs against the components (receptor and ligands) of the human insulin-like growth factor (IGF) system were selected from the new library that could not be selected from the previously constructed library. The newly identified dAbs were highly soluble, expressible, monomeric and may have potential as candidate cancer therapeutics. 5) We have continued to characterize our previously identified human mAbs against mesothelin and CD22. We showed their high activity in vitro. Further studies are needed to find whether they could be used as candidate therapeutics. 6) We constructed human anti HER2 single chain variable fragments (scFvs) with a C-terminal fusion polypeptide containing 1, 3, or 17 threonine (Thr) residues. The C-terminal extended fusion polypeptides of these recombinant scFv fusion proteins were used as acceptor substrates for human polypeptide-alpha-Nu-acetylgalactosaminyltransferase II (h-ppGalNAc-T2) that transfers either GalNAc or 2-keto-Gal, a modified galactose with a chemical handle, from their respective UDP-sugars to the side-chain hydroxyl group of the Thr residue(s). These fusion scFv proteins with the modified galactose were then conjugated with a fluorescence probe, Alexa488, that carries an orthogonal reactive group. The fluorescence labeled scFv proteins bound specifically to a human breast cancer cell line (SK-BR-3) that overexpresses HER2, indicating that the in vitro folded scFv fusion proteins are biologically active and the presence of conjugated multiple Alexa488 probes in their C-terminal end does not interfere with their binding to the antigen. This novel method for conjugation to specific sites could be used for construction of targeted nanoparticles guided by antibodies. 7) We have hypothesized that coupling of the current treatment modalities with a nano-drug delivery vehicle (immunoliposomes) could significantly improve the drug delivery efficiency thereby achieving better treatment efficacies. To test this hypothesis we generated a CD22 specific scFv which has an engineered cysteine at the C-terminus, conjugated it to liposomes and targeted these liposomes to CD22-expressing cells. We found that the targeted liposomes recognize specifically CD22 on BJAB cells but does not bind to cells that do not express CD22. Intracellular localization of the targeted liposomes at 370C but not at 40C (observed by confocal microscopy) indicated that our targeted liposomes were taken up through an energy dependent process via receptor-mediated endocytosis. Therefore, these 2nd-generation liposomes may serve as promising carriers for targeted delivery of anticancer agents to treat patients suffering from B-cell lymphoma. 8) We initiated a high-throughput sequencing of large portions of the antibody repertoires of humans (the human antibodyome). We believe that knowledge of the complete antibodyome will have implications for research, diagnosis, prevention and treatment of cancer. It can help for deeper understanding of the B cell (system) biology and diseases, help develop new diagnostic methods based on individual antibodyomes, help predict individual immune responses to immunization and therapeutics as well as provide information for the design of novel therapeutics. Most implications can not be predicted due to the very nature of an omic science.